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An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63

Nature Cell Biology volume 11, pages 278–285 (2009)Cite this article

Abstract

Activation of the protein kinases ATM and ATR following chromosomal breakage prevents initiation of DNA replication and entry into mitosis. However, the effects of ATM and ATR activation in cells already progressing through mitosis are poorly understood. Here we report that ATM and ATR activation induced by DNA double-strand breaks (DSBs) inhibits centrosome-driven spindle assembly in Xenopus laevis mitotic egg extract and somatic cells, delaying mitotic progression. Using a cDNA expression library to screen for ATM and ATR substrates, we identified centrosomal protein CEP63 as an ATM and ATR target required for normal spindle assembly. ATM and ATR phosphorylate Xenopus CEP63 (XCEP63) on Ser 560 and promote its delocalization from the centrosome. Suppression of ATM and ATR activity or mutation of XCEP63 Ser 560 to Ala prevented spindle assembly defects. Consistently, inactivation of the CEP63 gene in avian DT40 cells impaired spindle assembly and prevented ATM- and ATR-dependent effects on mitosis. These data indicate that ATM and ATR control mitotic events in vertebrate cells by targeting CEP63 and centrosome dependent spindle assembly.

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Figure 1: Effects of ATM and ATR activation on spindle assembly.
Figure 2: Effects of ATM and ATR activation on mitotic kinases and anastral spindles.
Figure 3: Identification and characterization of the ATM and ATR target XCEP63.
Figure 4: The function of XCEP63 in spindle assembly and in the ATM- and ATR-dependent checkpoint.
Figure 5: Characterization of CEP63 in DT40 cells.

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References

  1. Maller, J. L. et al. The mechanism of CSF arrest in vertebrate oocytes. Mol. Cell Endocrinol. 187, 173–178 (2002).

    Article  CAS  Google Scholar 

  2. Desai, A., Murray, A., Mitchison, T. J. & Walczak, C. E. The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro. Methods Cell Biol. 61, 385–412 (1999).

    Article  CAS  Google Scholar 

  3. Costanzo, V., Paull, T., Gottesman, M. & Gautier, J. Mre11 assembles linear DNA fragments into DNA damage signaling complexes. PLoS Biol. 2, E110 (2004).

    Article  Google Scholar 

  4. Yoo, H. Y., Shevchenko, A. & Dunphy, W. G. Mcm2 is a direct substrate of ATM and ATR during DNA damage and DNA replication checkpoint responses. J. Biol. Chem. 279, 53353–53364 (2004).

    Article  CAS  Google Scholar 

  5. Hickson, I. et al. Identification and characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM. Cancer Res. 64, 9152–9159 (2004).

    Article  CAS  Google Scholar 

  6. Jazayeri, A. et al. ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks. Nature Cell Biol. 8, 37–45 (2006).

    Article  CAS  Google Scholar 

  7. Potapova, T. A. et al. The reversibility of mitotic exit in vertebrate cells. Nature 440, 954–958 (2006).

    Article  CAS  Google Scholar 

  8. Liu, J. & Maller, J. L. Xenopus Polo-like kinase Plx1: a multifunctional mitotic kinase. Oncogene 24, 238–247 (2005).

    Article  CAS  Google Scholar 

  9. Sancar, A., Lindsey-Boltz, L. A., Unsal-Kacmaz, K. & Linn., S. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu. Rev. Biochem. 73, 39–85 (2004).

    Article  CAS  Google Scholar 

  10. Carazo-Salas, R. E. et al. Generation of GTP-bound Ran by RCC1 is required for chromatin-induced mitotic spindle formation. Nature 400, 178–181 (1999).

    Article  CAS  Google Scholar 

  11. Heald, R. et al. Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts. Nature 382, 420–425 (1996).

    Article  CAS  Google Scholar 

  12. Karsenti, E. & Vernos, I. The mitotic spindle: a self-made machine. Science 294, 543–547 (2001).

    Article  CAS  Google Scholar 

  13. Heald, R., Tournebize, R., Habermann, A., Karsenti, E. & Hyman, A. Spindle assembly in Xenopus egg extracts: respective roles of centrosomes and microtubule self-organization. J. Cell Biol. 138, 615–628 (1997).

    Article  CAS  Google Scholar 

  14. Lustig, K. D. et al. Small pool expression screening: identification of genes involved in cell cycle control, apoptosis, and early development. Methods Enzymol. 283, 83–99 (1997).

    Article  CAS  Google Scholar 

  15. Andersen, J. S. et al. Proteomic characterization of the human centrosome by protein correlation profiling. Nature 426, 570–574 (2003).

    Article  CAS  Google Scholar 

  16. Zachos, G. et al. Chk1 is required for spindle checkpoint function. Dev. Cell 12, 247–260 (2007).

    Article  CAS  Google Scholar 

  17. Matsumoto, Y. & Maller, J. L. Calcium, calmodulin and CaMKII requirement for initiation of centrosome duplication in Xenopus egg extracts. Science 295, 499–502 (2002).

    Article  CAS  Google Scholar 

  18. Dodson, H. et al. Centrosome amplification induced by DNA damage occurs during a prolonged G2 phase and involves ATM. EMBO J. 23, 3864–3873 (2004).

    Article  CAS  Google Scholar 

  19. Khodjakov, A., Cole, R. W., Oakley, B. R. & Rieder, C. L. Centrosome-independent mitotic spindle formation in vertebrates. Curr. Biol. 10, 59–67 (2000).

    Article  CAS  Google Scholar 

  20. Zachos, G., Rainey, M. D. & Gillespie, D. A. Chk1-deficient tumour cells are viable but exhibit multiple checkpoint and survival defects. EMBO J. 22, 713–723 (2003).

    Article  CAS  Google Scholar 

  21. Matsumoto, Y. & Maller, J. L. A centrosomal localization signal in cyclin E required for Cdk2-independent S. phase entry. Science 306, 885–888 (2004).

    Article  CAS  Google Scholar 

  22. Vagnarelli, P. et al. Condensin and Repo-Man-PP1 cooperate in the regulation of chromosome architecture during mitosis. Nature Cell Biol. 8, 1133–1142 (2006).

    Article  CAS  Google Scholar 

  23. Skoufias, D. A., Lacroix, F. B., Andreassen, P. R., Wilson, L. & Margolis, R. L. Inhibition of DNA decatenation, but not DNA damage, arrests cells at metaphase. Mol. Cell 15, 977–990 (2004).

    Article  CAS  Google Scholar 

  24. Goodarzi, A. A. et al. ATM signaling facilitates repair of DNA double-strand breaks associated with heterochromatin. Mol. Cell 31, 167–177 (2008).

    Article  CAS  Google Scholar 

  25. Morrison, C. & Rieder, C. L. Chromosome damage and progression into and through mitosis in vertebrates. DNA Repair 3, 1133–1139 (2004).

    Article  CAS  Google Scholar 

  26. Basto, R. et al. Centrosome amplification can initiate tumorigenesis in flies. Cell 133, 1032–1042 (2008).

    Article  CAS  Google Scholar 

  27. Griffith, E. et al. Mutations in pericentrin cause Seckel syndrome with defective ATR-dependent DNA damage signaling. Nature Genet. 40, 232–236 (2008).

    Article  CAS  Google Scholar 

  28. Buim, M. E., Soares, F. A., Sarkis, A. S. & Nagai, M. A. The transcripts of SFRP1, CEP63 and EIF4G2 genes are frequently downregulated in transitional cell carcinomas of the bladder. Oncology 69, 445–454 (2005).

    Article  Google Scholar 

  29. Morris, J. A., Kandpal, G., Ma, L. & Austin, C. P. DISC1 (disrupted-in-schizophrenia 1) is a centrosome-associated protein that interacts with MAP1A, MIPT3, ATF4/5 and NUDEL: regulation and loss of interaction with mutation. Hum. Mol. Genet. 12, 1591–1608 (2003).

    Article  CAS  Google Scholar 

  30. Kamiya, A. et al. A schizophrenia-associated mutation of DISC1 perturbs cerebral cortex development. Nature Cell Biol. 7, 1167–1178 (2005).

    Article  Google Scholar 

Download references

Acknowledgements

We thank Tim Hunt, members of Clare Hall Laboratories and of the Genome Stability Unit for their comments. We thank H Mahbubani and J Kirk for technical support with Xenopus laevis. This work was funded by Cancer Research UK. V. Costanzo is also supported by the Lister Institute of Preventive Medicine, the European Research Council (ERC) start up grant and the EMBO Young Investigator Program (YIP). A. Vindigni thanks AIRC for its support.

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Authors and Affiliations

  1. Genome Stability Unit, London Research Institute, Clare Hall Laboratories, South Mimms, London, EN6 3LD, UK

    Eloise Smith, Donniphat Dejsuphong, Alessia Balestrini & Vincenzo Costanzo

  2. Department of Radiation Genetics, Kyoto University Graduate, School of Medicine, Yoshidakonoe, Sakyo-ku, Kyoto, 606-8501, Japan

    Donniphat Dejsuphong & Shunichi Takeda

  3. ICGEB (International Centre for Genetic Engineering and Biotechnology) Padriciano, Trieste, 9934012, Italy

    Martin Hampel & Alessandro Vindigni

  4. Applied Biosystems, Frankfurter Darmstadt, Strasse 129 B, 64293, Germany

    Christof Lenz

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Contributions

E.S, D.D. and A.B. performed the experiments and analysed the data in the Xenopus system and in avian DT40 cells; M.H., C.L. and A.V. provided technical and conceptual advice with mass spectrometry; S.T. provided technical and conceptual advice with avian DT40 cells; V.C. planned the experiments and wrote the manuscript.

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Correspondence to Vincenzo Costanzo.

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Smith, E., Dejsuphong, D., Balestrini, A. et al. An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63. Nat Cell Biol 11, 278–285 (2009). https://doi.org/10.1038/ncb1835

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